Composition for inhibition of insect host sensing

ABSTRACT

The present invention provide chemical modulators of insect olfactory receptors. In particular, compounds and compositions are provided that can inhibit host targeting functions in insects such as mosquitos. Method of employing such agents, and articles incorporating the same, are also provided.

This application claims benefit of priority to U.S. ProvisionalApplication Ser. No. 61/406,368, filed Oct. 25, 2010, and 61/406,786,filed Oct. 26, 2010, the entire contents of which are herebyincorporated by reference.

BACKGROUND OF THE INVENTION I. Field of the Invention

The present invention relates to the fields of entomology and infectiousdisease. More particular, the invention relates to methods andcompositions for disrupting host-targeting in mosquitoes and otherinsects.

II. Related Art

Insects acting as agricultural pests and disease vectors are responsiblefor extraordinary economic and medical impacts, respectively. Humanmalaria affects regions that are home to over two billion people, andcause at least one million deaths each year. The social and economicimpact of the disease are staggering, with a disproportionate number ofdeaths coming in children aged 5 or less. And despite successes inlimiting the disease in the last half of the previous century, recenttrends show a resurgence in malarial infections in certain areas, andsuggest a shift in modes of malarial transmission.

Currently the primary tool to prevent the spread of malaria is the useof insecticides that kill the mosquito vector. However, each of thevarious forms of insecticide treatment—residual house spraying,insecticide treated clothes, bedding and netting, and chemicallarviciding—have drawbacks, including environmental and host toxicity,limited duration and need for insect contact. Biological larviciding canavoid toxicity issues, but takes time and is quite expensive.Chemoprophylaxis is also expensive and may have unacceptable sideeffects. Finally, segregating populations is expensive and in many cases(developing world countries) impractical.

Thus, while there are many different ways to attack malaria, and eachhave contributed substantially to limiting the spread of disease, theyalso each have limitations that leave room for substantial improvement.

SUMMARY OF THE INVENTION

Thus, in accordance with the present invention, there is provided amethod of disrupting host-seeking behavior in an insect comprisingproviding to an insect environment a compound that binds to and/oragonizes, insect Orco ion channels (previously designated AgOR7 inAnopheles gambiae, but referred herein as equivalents). The insect maybe a Culcine or Anophelin mosquito, a Dipteran, Lepidopteran or Ixodidainsect. Other agricultural pest insects include Acalymma, Aclerisvariegana, African armyworm, Africanized bee, Agromyzidae, Agrotismunda, Agrotis porphyricollis, Aleurocanthus woglumi, Aleyrodesproletella, Anasa tristis, Anisoplia austriaca, Anthonomus pomorum,Anthonomus signatus, Aonidiella aurantii, Aphid, Aphis fabae, Aphisgossypii, Apple maggot, Argentine ant, Army cutworm, Arotrophoraarcuatalis, Asterolecanium coffeae, Australian plague locust,Bactericera cockerelli, Bactrocera, Bactrocera correcta, Bagradahilaris, Banded hickory borer, Banksia Boring Moth, Beet armyworm,Bogong moth, Boll weevil, Brevicoryne brassicae, Brown locust, Brownmarmorated stink bug, Brown planthopper, Cabbage Moth, Cabbage worm,Callosobruchus maculates, Cane beetle, Carrot fly, Cecidomyiidae,Ceratitis capitata, Cereal leaf beetle, Chlorops pumilionis, Citruslong-horned beetle, Coccus viridis, Codling moth, Coffee borer beetle,Colorado potato beetle, Confused flour beetle, Crambus, Cucumber beetle,Curculio nucum, Dark Sword-grass, Date stone beetle, Delia (genus),Delia antique, Delia floralis, Delia radicum, Desert locust, Diabrotica,Diamondback moth, Diaphania indica, Diaphania nitidalis, Diaphorinacitri, Diaprepes abbreviates, Differential grasshopper, Dociostaurusmaroccanus, Drosophila suzukii, Erionota thrax, Eriosomatinae,Eumetopina flavipes, European Corn Borer, Eurydema oleracea, Eurygasterintegriceps, Forest bug, Frankliniella occidentalis, Fankliniellatritici, Galleria mellonella, Garden Dart, Greenhouse whitefly,Gryllotalpa orientalis, Gryllus pennsylvanicus, Gpsy moths, Helicoverpaarmigera, Helicoverpa zea, Henosepilachna vigintioctopunctata, Hessianfly, Japanese beetle, Khapra beetle, Lampides boeticus, Leaf miner,Lepidiota consobrina, Lepidosaphes ulmi, Leptoglossus zonatus,Leptopterna dolabrata, Lesser wax moth, Leucoptera (moth), Leucopteracaffeine, Light brown apple moth, Lissorhoptrus oryzophilus, Long-tailedSkipper, Lygus (genus), Lygus hesperus, Maconellicoccus hirsutus,Macrodactylus subspinosus, Macrosiphum euphorbiae, Maize weevil, Manducasexta, Mayetiola hordei, Mealybug, Megacopta cribraria, Moth, Leek moth,Myzus persicae, Nezara viridula, Olive fruit fly, Opomyzidae, Papiliodemodocus, Paracoccus marginatus, Paratachardina pseudolobata, Peaaphid, Pentatomoidea, Phthorimaea operculella, Phyllophaga (genus),Phylloxera, Phylloxeroidea, Pieris brassicae, Pink bollworm, Platynotaidaeusalis, Plum curculio, Pseudococcus viburni, Pyralis farinalis, Redimported fire ant, Red locust, Rhagoletis cerasi, Rhagoletisindifferens, Rhagoletis mendax, Rhopalosiphum maidis, Rhynchophorusferrugineus, Rhyzopertha dominica, Rice Moth, Russian wheat aphid, SanJose scale, Sciaridae, Scirtothrips dorsalis, Scutelleridae, Serpentineleaf miner, Silver Y, Silverleaf whitefly, Small hive beetle, Soybeanaphid, Spodoptera cilium, Spodoptera litura, Spotted cucumber beetle,Squash vine borer, Stenotus binotatus, Sternorrhyncha, Strauzialongipennis, Striped flea beetle, Sunn pest, Sweetpotato bug, Tarnishedplant bug, Thrips (genus), Thrips palmi, Toxoptera citricida, Triozaerytreae, Tuta absoluta, Varied carpet beetle, Virachola Isocrates,Waxworm, Western corn rootworm, Wheat fly, Wheat weevil, Winter Moth,and Xyleborus glabratus.

The method may comprise contacting a host surface located in saidenvironment with said compound; may comprise aerosol or mist delivery tosaid environment; may comprise application to a water surface in saidenvironment; may comprise application to a shelter or clothing surfacein said environment; may comprise use of a shelter or article ofclothing containing said compound in said environment; or may comprisedepositing a solid form of said compound in said environment. Thecompound may be VUAA1. The method may further comprise provision of aninsect repellent to said environment, such as a mosquito repellent.

In another embodiment, there is provided a container comprising a VUAA1in a liquid or gaseous dispersion. The gaseous dispersion may be anaerosol. The container may further comprising a nozzle or valve, aporous applicator, or a rolling applicator.

In yet another embodiment, there is provided a fabric or materialcomprising VUAA1. The fabric or material may clothing, a shelter,bedding or netting.

In still another embodiment, there is provided a water soluble tabletcomprising VUAA1. Further embodiments include:

-   -   methods of disrupting transmission of a mosquito-borne disease        comprising providing to a mosquito environment a compound that        binds to and/or agonizes a mosquito ORco ion channel, such as        AgOR7;    -   methods of reducing mosquito bites comprising providing to a        mosquito environment a compound that binds to and/or agonizes a        mosquito ORco ion channel, such as AgOR7;    -   methods of reducing mosquito reproduction comprising providing        to a mosquito environment a compound that binds to and/or        agonizes a mosquito ORco ion channel, such as AgOR7;    -   methods of reducing mosquito infestation in an environment        comprising providing to said environment a compound that binds        to and/or agonizes a mosquito ORco ion channel, such as AgOR7;    -   methods of reducing crop damage by an insect population        comprising providing to a crop-growing environment a compound        that binds to and/or agonizes, an ORco ion channel; and    -   a method of reducing infestation of a crop-damaging insect        comprising providing to a crop-growing environment populated by        a a crop-damaging insect population a compound that binds to        and/or agonizes, an ORco ion channel.

It is contemplated that any method or composition described herein canbe implemented with respect to any other method or composition describedherein.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

These, and other, embodiments of the invention will be betterappreciated and understood when considered in conjunction with thefollowing description and the accompanying drawings. It should beunderstood, however, that the following description, while indicatingvarious embodiments of the invention and numerous specific detailsthereof, is given by way of illustration and not of limitation. Manysubstitutions, modifications, additions and/or rearrangements may bemade within the scope of the invention without departing from the spiritthereof, and the invention includes all such substitutions,modifications, additions and/or rearrangements.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Olfactory cues make up the principal sensory modalities inmediating several key behaviors in adult mosquitoes. These includenectar feeding, selection of oviposition sites, mate selection andespecially host (blood-meal) preference where chemical and temperatureinputs synergize most.

FIG. 2. Canonical and Non-Canonical Models of General Insect OlfactorySignal Transduction. Schematic incorporating recent insights intomolecular interactions in the lumen and dendritic membrane of insectORNs. General odorants entering through cuticular pores are loaded ontoOBPs that facilitate transport to conventional ORs (ORx) within thecontext of canonical OR complexes. Transport of odorants is directed bya specific OBP which may physically interact with the conventionaland/or Orco family OR. Pheromone-sensitive pathways are likely toinvolve additional molecular components. In canonical models (I, II),conventional ORs (OrX) bind odorants and physically interact with highlyconserved, non-conventional Orco family ORs (AgO7 in An. gambiae) toform functional heteromultimers expressed in a majority of ORNs. In thismodel, binding of odorants activate ionotropic (Sato et al., 2008) and,possibly, metabotropic (Wicher et al., 2008) signaling pathways. Inother ORNs, non-canonical ORs (III), such as members of the IGluR/IRgene family (Benton et al., 2009) respond to atypical odorant that insome cases (e.g., ammonia and lactic acid) are associated withhuman-derived kairomones.

FIGS. 3A-F. VUAA1 evokes macroscopic currents in HEK293 cells expressingAgORco and its orthologs. (FIG. 3A) Structure of VUAA1. (FIG. 3B)Concentration response curves (CRCs) generated from Fluo-4 acetoxymethylester-based Ca²⁺ imaging with AgORco and AgORco+AgOR10, cell lines inresponse to VUAA1. (FIGS. 3C-D) Whole-cell patch clamp recordings ofconcentration-dependent responses to VUAA1 in cells stably expressingAgORco alone and AgORco+AgOR10. (FIG. 3E) Benzaldehyde (BA), an AgOR10agonist, elicits concentration-dependent responses to AgORco+AgOR10cells. (FIG. 3F) Whole-cell current responses to VUAA1 in HEK293 cellsexpressing DmOR83b and HvOR2. Cells in FIGS. 3C, 3D, 3E, and 3F wereclamped at −60 mV.

FIGS. 4A-D. Channel-like currents result from application of VUAA1 tocells expressing AgORco alone or in complex. (FIGS. 4A-C),Representative traces of voltage-dependent currents in AgORco (FIG. 4A)and AgORco+AgOR10 (FIGS. 4B-C) cells. Holding potentials ranged from −60mV to +40 mV in 20 mV increments. (FIG. 4D), Current-voltagerelationships of (FIG. 4A) n=3, (FIG. 4B) n=7, and (FIG. 4C) n=4 fromnormalized peak currents.

FIGS. 5A-D. AgORco is a functional channel and responds to VUAA1 inoutside-out membrane patches. (FIG. 5A) Single-channel recording from anoutside-out excised patch pulled from an HEK293 cell-expressing AgORco7.(FIGS. 5B-D) Expansions of trace (FIG. 5A) before (FIG. 5B) during (FIG.5C), and after (FIG. 5D) a 5 s application of −4.0 log M VUAA1.All-point current histograms of trace expansions are inset in FIGS.5B-D. Excised membrane patch was held at −60 mV.

FIGS. 6A-D. VUAA1 activates AgORco-expressing neurons in Anophelesgambiae females. (FIG. 6A) Representative traces of SSR recordings fromcapitate peg sensilla upon electrode puncture. VUAA1 or vehicle alone(DMSO) was delivered through the glass recording electrode. CpA isdiscernible from the smaller CpB/C action potentials. Preparations werekept under a steady stream of humidified, synthetic air (21% O₂/79% N₂)to limit the basal activity of CpA. (FIG. 6B) Expansions of traces as inFIG. 3A. (FIG. 6C) Activity of CpA neuron in response to VUAA1. Spikefrequency was calculated every second for the first 10 s after sensillumpuncture and every 10 s thereafter. After 60 s, the preparation waspulsed for 2 s with atmospheric air to confirm a functional CpA neuron.Sensilla that did not respond to CO2 or 1-octen-3-ol were excluded fromanalysis. (FIG. 6D) Activity of CpB/CpC neurons in response to VUAA1 asin FIG. 6C.

FIGS. 7A-F. VUAA1 and BA responses are OR specific. (FIG. 7A) Histogramof normalized currents from concentration-dependent responses in FIGS.7C-E (n=5). (FIG. 7B) Un-transfected HEK293 cells did not respond toeither VUAA1 or BA (n=5). (FIG. 7C) GFP was co-transfected with DmOR83bor HvOR2 to identify cells expressing the OR. GFP alone cells had nocurrents from VUAA1 or BA (n=4). (FIGS. 7D-E) For comparison, AgORco andAgORco+AgOR10 cells both depolarized during VUAA1 application, whileonly AgORco+AgOR10 cells responded to BA. Holding potentials for allrecordings were −60 mV. (FIG. 7F) VUAA1 did not elicit currents in cellsstably expressing another cation channel, rat transient receptorpotential vanilloid 1 (rTRPV1), but did respond to the agonistcapsaicin.

FIGS. 8A-D. 8-Br-cAMP and 8-Br-cGMP did not elicit currents in AgORco orAgORco+AgOR10 cells. (FIG. 8A) Representative trace of whole-cellrecordings from cells expressing AgORco+AgOR10 with application of8-Br-cAMP, 8-Br-cGMP, and BA (n=4). (FIG. 8B) Representative trace fromAgORco cells with application of 8-Br-cAMP, 8-Br-cGMP, and VUAA1 (n=4).Holding potentials for all recordings were −60 mV. (FIG. 8C)Representative trace from cells expressing rCNGA2 with application of8-Br-cGMP. Holding potentials for all recording were −60 mV. (FIG. 8D)Histogram of normalized currents from cyclic nucleotide and controlresponses (n=5).

FIG. 9. VUAA1's larval activity threshold relative to DEET is 5 ordersof magnitude lower and AgORco dependent. Larval activity in An. gambiaeover 5 minutes monitored in 6-well culture plates using Ethovision(Noldus) software. Larval responses to VUAA1 (third through eighth fromleft) are significantly higher than those to DEET (ninth througheleventh from left). Gene silencing studies (twelfth through fourteenthfrom left) demonstrate that larval responses to VUAA1 areAgORco-dependent in keeping with VUAA1's mode of action.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Insects interpret their chemical environment through the use of a familyof cell-surface odorant receptors (ORs) to sense volatile chemicalsknown as odorants. For odorant reception to take place, a member of theORco family of ORs must be present to couple to another highly diverseOR (ORX) that is responsible for sensing different odors. Each insectspecies has many Ors that generally species-specific, but only one Orcofamily member that are extremely conserved throughout all insect taxa.There have been no reported ORco ligands to date. As part of aHigh-Throughput Screen to identify compounds that modulate OR activity,the present inventors have discovered the first ORco family activator.This ORco family activator, termed VUAA1, has the theoretical ability toactivate all ORX/ORco complexes. The host-seeking behavior ofblood-feeding insects is principally driven through their sense ofsmell. This blood-feeding behavior serves as the foundation for theirability to transit disease. The capacity to disrupt olfactory-mediatedbehavior through direct chemical interference, as by VUAA1, would be amajor advance in the fight against vector-borne diseases, and modulationof the ORco complex would render the insect incapable of performing itsusual behaviors, such as host-seeking and nectar feeding. VUAA1 can alsobe used to disrupt the behavior of agriculturally important insect pestsresponsible for billions of dollars of crop damage worldwide each year.

I. MOSQUITOES

Mosquito, from the Spanish or Portuguese meaning “little fly,” is acommon insect in the family Culicidae. Mosquitoes resemble crane flies(family Tipulidae) and chironomid flies (family Chironomidae), withwhich they are sometimes confused by the casual observer.

Mosquitoes go through four stages in their life-cycle: egg, larva, pupa,and adult or imago. Adult females lay their eggs in water, which can bea salt-marsh, a lake, a puddle, a natural reservoir on a plant, or anartificial water container such as a plastic bucket. The first threestages are aquatic and last 5-14 days, depending on the species and theambient temperature; eggs hatch to become larvae, then pupae. The adultmosquito emerges from the pupa as it floats at the water surface. Adultslive for 4-8 weeks.

Female mosquitoes have mouthparts that are adapted for piercing the skinof plants and animals. While males typically feed on nectar and plantjuices, the female needs to obtain nutrients from a “blood meal” beforeshe can produce eggs.

Mosquito larvae have a well-developed head with mouth brushes used forfeeding, a large thorax with no legs and a segmented abdomen. Larvaebreathe through spiracles located on the eighth abdominal segment, orthrough a siphon, and therefore must come to the surface frequently. Thelarvae spend most of their time feeding on algae, bacteria, and othermicro-organisms in the surface microlayer. They dive below the surfaceonly when disturbed. Larvae swim either through propulsion with themouth brushes, or by jerky movements of the entire body. Larvae developthrough four stages, or instars, after which they metamorphose intopupae. At the end of each instar, the larvae molt, shedding theirexoskeleton, or skin, to allow for further growth. Length of the adultvaries but is rarely greater than 16 mm (0.6 in), and weight up to 2.5mg (0.04 grain). All mosquitoes have slender bodies with three sections:head, thorax and abdomen.

The pupa is comma-shaped, as in Anopheles when viewed from the side. Thehead and thorax are merged into a cephalothorax with the abdomencircling around underneath. As with the larvae, pupae must come to thesurface frequently to breathe, which they do through a pair ofrespiratory trumpets on the cephalothorax. However, pupae do not feedduring this stage. After a few days, the dorsal surface of thecephalothorax splits and the adult mosquito emerges. The pupa is lessactive than larva.

The duration from egg to adult varies among species and is stronglyinfluenced by ambient temperature. Mosquitoes can develop from egg toadult in as little as five days but usually take 10-14 days in tropicalconditions. The variation of the body size in adult mosquitoes dependson the density of the larval population and food supply within thebreeding water. Adult flying mosquitoes frequently rest in a tunnel thatthey build right below the roots of the grass.

Adult mosquitoes usually mate within a few days after emerging from thepupal stage. In most species, the males form large swarms, usuallyaround dusk, and the females fly into the swarms to mate.

Males live for about a week, feeding on nectar and other sources ofsugar. Females will also feed on sugar sources for energy but usuallyrequire a blood meal for the development of eggs. After obtaining a fullblood meal, the female will rest for a few days while the blood isdigested and eggs are developed. This process depends on the temperaturebut usually takes 2-3 days in tropical conditions. Once the eggs arefully developed, the female lays them and resumes host seeking. Thecycle repeats itself until the female dies. Their lifespan depends ontemperature, humidity, and also their ability to successfully obtain ablood meal while avoiding host defenses.

The head is specialized for acquiring sensory information and forfeeding. The head contains the eyes and a pair of long, many-segmentedantennae. The antennae are important for detecting host odors as well asodors of breeding sites where females lay eggs. In all mosquito species,the antennae of the males in comparison to the females are noticeablybushier and contain auditory receptors to detect the characteristicwhine of the female. The compound eyes are distinctly separated from oneanother. Their larvae only possess a pit-eye ocellus. The compound eyesof adults develop in a separate region of the head. New ommatidia areadded in semicircular rows at the rear of the eye; during the firstphase of growth, this leads to individual ommatidia being square, butlater in development they become hexagonal. The hexagonal pattern willonly become visible when the carapace of the stage with square eyes ismolted. The head also has an elongated, forward-projecting“stinger-like” proboscis used for feeding, and two sensory palps. Themaxillary palps of the males are longer than their proboscis whereas thefemales' maxillary palps are much shorter. As with many members of themosquito family, the female is equipped with an elongated proboscis thatshe uses to collect blood to feed her eggs.

The thorax is specialized for locomotion. Three pairs of legs and a pairof wings are attached to the thorax. The insect wing is an outgrowth ofthe exoskeleton. The Anopheles mosquito can fly for up to four hourscontinuously at 1 to 2 kilometres per hour (0.62 to 1.2 mph) travellingup to 12 km (7.5 mi) in a night.

The abdomen is specialized for food digestion and egg development. Thissegmented body part expands considerably when a female takes a bloodmeal. The blood is digested over time serving as a source of protein forthe production of eggs, which gradually fill the abdomen.

The duration from egg to adult varies among species and is stronglyinfluenced by ambient temperature. Mosquitoes can develop from egg toadult in as little as five days but usually take 10-14 days in tropicalconditions. The variation of the body size in adult mosquitoes dependson the density of the larval population and food supply within thebreeding water. Adult flying mosquitoes frequently rest in a tunnel thatthey build right below the roots of the grass.

Adult mosquitoes usually mate within a few days after emerging from thepupal stage. In most species, the males form large swarms, usuallyaround dusk, and the females fly into the swarms to mate. Males live forabout a week, feeding on nectar and other sources of sugar. Females willalso feed on sugar sources for energy but usually require a blood mealfor the development of eggs. After obtaining a full blood meal, thefemale will rest for a few days while the blood is digested and eggs aredeveloped. This process depends on the temperature but usually takes 2-3days in tropical conditions. Once the eggs are fully developed, thefemale lays them and resumes host seeking. The cycle repeats itselfuntil the female dies. Their lifespan depends on temperature, humidity,and also their ability to successfully obtain a blood meal whileavoiding host defenses.

In order for the mosquito to obtain a blood meal it must circumvent thevertebrate physiological responses. The mosquito, as with allblood-feeding arthropods, has mechanisms to effectively block thehemostasis system with their saliva, which contains a mixture ofsecreted proteins. Mosquito saliva negatively affects vascularconstriction, blood clotting, platelet aggregation, angiogenesis andimmunity and creates inflammation. Universally, hematophagous arthropodsaliva contains at least one anticlotting, one anti-platelet, and onevasodilatory substance. Mosquito saliva also contains enzymes that aidin sugar feeding and antimicrobial agents to control bacterial growth inthe sugar meal. The composition of mosquito saliva is relatively simpleas it usually contains fewer than 20 dominant proteins. Despite thegreat strides in knowledge of these molecules and their role inbloodfeeding achieved recently, scientists still cannot ascribefunctions to more than half of the molecules found in arthropod saliva.One promising application is the development of anti-clotting drugsbased on saliva molecules, which might be useful for approachingheart-related disease, because they are more user-friendly bloodclotting inhibitors and capillary dilators.

Two important events in the life of female mosquitoes are eggdevelopment and blood digestion. After taking a blood meal the midgut ofthe female synthesizes proteolytic enzymes that hydrolyze the bloodproteins into free amino acids. These are used as building blocks forthe synthesis of egg yolk proteins.

II. MOSQUITO-BORNE DISEASE

Mosquitoes are a vector agent that carries disease-causing viruses andparasites from person to person without catching the disease themselves.The principal mosquito borne diseases are the viral diseases yellowfever, dengue fever and Chikungunya, transmitted mostly by the Aedesaegypti, and malaria carried by the genus Anopheles. Though originally apublic health concern, HIV is now thought to be almost impossible formosquitoes to transmit.

Mosquitoes are estimated to transmit disease to more than 700 millionpeople annually in Africa, South America, Central America, Mexico andmuch of Asia with millions of resulting deaths. At least 2 millionpeople annually die of these diseases.

Methods used to prevent the spread of disease, or to protect individualsin areas where disease is endemic include vector control aimed atmosquito eradication, disease prevention, using prophylactic drugs anddeveloping vaccines and prevention of mosquito bites, with insecticides,nets and repellents. Since most such diseases are carried by “elderly”females, scientists have suggested focusing on these to avoid theevolution of resistance.

A. Protozoa

The mosquito genus Anopheles carries the malaria parasite (seePlasmodium). Worldwide, malaria is a leading cause of prematuremortality, particularly in children under the age of five. It iswidespread in tropical and subtropical regions, including parts of theAmericas (22 countries), Asia, and Africa. Each year, there areapproximately 350-500 million cases of malaria, killing between one andthree million people, the majority of whom are young children insub-Saharan Africa. Ninety percent of malaria-related deaths occur insub-Saharan Africa. Malaria is commonly associated with poverty, and canindeed be a cause of poverty and a major hindrance to economicdevelopment.

Five species of the plasmodium parasite can infect humans; the mostserious forms of the disease are caused by Plasmodium falciparum.Malaria caused by Plasmodium vivax, Plasmodium ovale and Plasmodiummalariae causes milder disease in humans that is not generally fatal. Afifth species, Plasmodium knowlesi, is a zoonosis that causes malaria inmacaques but can also infect humans.

Malaria is naturally transmitted by the bite of a female Anophelesmosquito. When a mosquito bites an infected person, a small amount ofblood is taken, which contains malaria parasites. These develop withinthe mosquito, and about one week later, when the mosquito takes its nextblood meal, the parasites are injected with the mosquito's saliva intothe person being bitten. After a period of between two weeks and severalmonths (occasionally years) spent in the liver, the malaria parasitesstart to multiply within red blood cells, causing symptoms that includefever, and headache. In severe cases the disease worsens leading tohallucinations, coma, and death.

A wide variety of antimalarial drugs are available to treat malaria. Inthe last 5 years, treatment of P. falciparum infections in endemiccountries has been transformed by the use of combinations of drugscontaining an artemisinin derivative. Severe malaria is treated withintravenous or intramuscular quinine or, increasingly, the artemisininderivative artesunate. Several drugs are also available to preventmalaria in travellers to malaria-endemic countries (prophylaxis).Resistance has developed to several antimalarial drugs, most notablychloroquine.

Malaria transmission can be reduced by preventing mosquito bites bydistribution of inexpensive mosquito nets and insect repellents, or bymosquito-control measures such as spraying insecticides inside housesand draining standing water where mosquitoes lay their eggs. Althoughmany are under development, the challenge of producing a widelyavailable vaccine that provides a high level of protection for asustained period is still to be met.

B. Helminthiasis

Some species of mosquito can carry the filariasis worm, a parasite thatcauses a disfiguring condition (often referred to as elephantiasis)characterized by a great swelling of several parts of the body;worldwide, around 40 million people are living with a filariasisdisability. The thread-like filarial nematodes (roundworms) are membersof the superfamily Filarioidea, also known as “filariae.” There are 9known filarial nematodes which use humans as the definitive host. Theseare divided into 3 groups according to the niche within the body thatthey occupy: lymphatic filariasis, subcutaneous filariasis, and serouscavity filariasis. Lymphatic filariasis is caused by the wormsWuchereria bancrofti, Brugia malayi, and Brugia timori. These wormsoccupy the lymphatic system, including the lymph nodes, and in chroniccases these worms lead to the disease elephantiasis. Subcutaneousfilariasis is caused by loa loa (the African eye worm), Mansonellastreptocerca, Onchocerca volvulus, and Dracunculus medinensis (theguinea worm). These worms occupy the subcutaneous layer of the skin, inthe fat layer. Serous cavity filariasis is caused by the wormsMansonella perstans and Mansonella ozzardi, which occupy the serouscavity of the abdomen. In all cases, the transmitting vectors are eitherblood sucking insects (flies or mosquitoes), or copepod crustaceans inthe case of Dracunculus medinensis.

Individuals infected by filarial worms may be described as either“microfilaraemic” or “amicrofilaraemic,” depending on whether or notmicrofilaria can be found in their peripheral blood. Filariasis isdiagnosed in microfilaraemic cases primarily through direct observationof microfilaria in the peripheral blood. Occult filariasis is diagnosedin amicrofilaraemic cases based on clinical observations and, in somecases, by finding a circulating antigen in the blood.

C. Viruses

The viral disease yellow fever, an acute hemorrhagic disease, istransmitted mostly by Aedes aegypti mosquitoes. The virus is a 40 to 50nm enveloped RNA virus with positive sense of the Flaviviridae family.The yellow fever virus is transmitted by the bite of female mosquitoes(the yellow fever mosquito, Aedes aegypti, and other species) and isfound in tropical and subtropical areas in South America and Africa, butnot in Asia. The only known hosts of the virus are primates and severalspecies of mosquito. The origin of the disease is most likely to beAfrica, from where it was introduced to South America through the slavetrade in the 16th century. Since the 17th century, several majorepidemics of the disease have been recorded in the Americas, Africa andEurope. In the 19th century, yellow fever was deemed one of the mostdangerous infectious diseases.

Clinically, yellow fever presents in most cases with fever, nausea, andpain and it generally subsides after several days. In some patients, atoxic phase follows, in which liver damage with jaundice (giving thename of the disease) can occur and lead to death. Because of theincreased bleeding tendency (bleeding diathesis), yellow fever belongsto the group of hemorrhagic fevers. The WHO estimates that yellow fevercauses 200,000 illnesses and 30,000 deaths every year in unvaccinatedpopulations; around 90% of the infections occur in Africa.

A safe and effective vaccine against yellow fever has existed since themiddle of the 20th century and some countries require vaccinations fortravelers. Since no therapy is known, vaccination programs are, alongwith measures to reduce the population of the transmitting mosquito, ofgreat importance in affected areas. Since the 1980s, the number of casesof yellow fever has been increasing, making it a reemerging disease.

Dengue fever and dengue hemorrhagic fever (DHF) are acute febrilediseases also transmitted by Aedes aegypti mosquitoes. These occur inthe tropics, can be life-threatening, and are caused by four closelyrelated virus serotypes of the genus Flavivirus, family Flaviviridae. Itis also known as breakbone fever, since it can be extremely painful. Itoccurs widely in the tropics, and increasingly in southern China. Unlikemalaria, dengue is just as prevalent in the urban districts of its rangeas in rural areas. Each serotype is sufficiently different that there isno cross-protection and epidemics caused by multiple serotypes(hyperendemicity) can occur. Dengue is transmitted to humans by theAedes (Stegomyia) aegypti or more rarely the Aedes albopictus mosquito.The mosquitoes that spread dengue usually bite at dusk and dawn but maybite at any time during the day, especially indoors, in shady areas, orwhen the weather is cloudy. The WHO says some 2.5 billion people, twofifths of the world's population, are now at risk from dengue andestimates that there may be 50 million cases of dengue infectionworldwide every year. The disease is now endemic in more than 100countries.

Other viral diseases like epidemic polyarthritis, Rift Valley fever,Ross River Fever, St. Louis encephalitis, West Nile virus (WNV),Japanese encephalitis, La Crosse encephalitis and several otherencephalitis type diseases are carried by several different mosquitoes.Eastern equine encephalitis (EEE) and Western equine encephalitis (WEE)occurs in the United States where it causes disease in humans, horses,and some bird species. Because of the high mortality rate, EEE and WEEare regarded as two of the most serious mosquito-borne diseases in theUnited States. Symptoms range from mild flu-like illness toencephalitis, coma and death. Culex and Culiseta are also involved inthe transmission of disease. WNV has recently been a concern in theUnited States, prompting aggressive mosquito control programs.

D. Transmission

A mosquito's period of feeding is often undetected; the bite onlybecomes apparent because of the immune reaction it provokes. When amosquito bites a human, she injects saliva and anti-coagulants. For anygiven individual, with the initial bite there is no reaction but withsubsequent bites the body's immune system develops antibodies and a bitebecomes inflamed and itchy within 24 hours. This is the usual reactionin young children. With more bites, the sensitivity of the human immunesystem increases, and an itchy red hive appears in minutes where theimmune response has broken capillary blood vessels and fluid hascollected under the skin. This type of reaction is common in olderchildren and adults. Some adults can become desensitized to mosquitoesand have little or no reaction to their bites, while others can becomehyper-sensitive with bites causing blistering, bruising, and largeinflammatory reactions, a response known as Skeeter Syndrome.

III. INSECT OLFACTORY RECEPTORS

The ability to detect and respond to the chemical environment iscritical sensory input into many essential behaviors of hematophagous(blood-feeding) insects (Zwiebel and Takken, 2004; FIG. 1). The searchfor vertebrate blood meals typically involves a flight of some distanceto reach the host. This behavior consists of a series of behavioralstages, beginning with the activation of a receptive insect by the hostchemical odor (kairomone) and ending when the insect alights on the host(Takken, 1991). At close range, attraction is mediated by severalodorants, one of which is CO₂. In combination with other host-derivedorganic chemicals, CO₂ acts as a synergist as it greatly enhances theattraction triggered by other volatiles (Gilles, 1980). Moreover, itappears that mosquitoes respond to changes in the concentration of CO2,rather than its presence or absence. In Ae. aegypti, changes in thefiring rate of CO₂ receptors have been observed with increases inconcentration of as little as 0.01% (Kellogg, 1970), while alterationsin behavior have been observed after increases of 0.03-0.05% (Eiras andJepson, 1991). Furthermore, a close examination of the role of CO₂revealed that the turbulence of the odor plume in the laboratory greatlyaffected the responsiveness of Ae. aegypti and An. gambiae s.s. (Dekkeret al., 2001a).

An. gambiae has also been shown to be attracted to acetone, lactic acid(Acree et al., 1968), carboxylic acids (Meijerink and van Loon, 1999),ammonia, 4-methyl-phenol, 1-octen-3-ol and other components of sweat(Cork and Park, 1996; Meijerink et al., 2001), as well as to the odor ofhuman feet, expired air and several unidentified components of Limburgercheese (De Jong and Knols, 1995). Furthermore, the often-citeddifferences in human attractiveness for mosquitoes (Curtis, 1986) isalmost certainly olfactory based (Qiu et al., 2006a; Schreck et al.,1990). This within-host differential behavior is most particularlyexpressed in anthropophilic culicids such as Ae. aegypti and An. gambiaes.s. (de Jong and Knols, 1995; Lindsay et al., 1993; Schreck et al.,1990). Host age but not gender may affect these inter-individualdifferences (Carnevale et al., 1978); race also appears to have noeffect (Schreck et al., 1990). Young children have been shown to be lessattractive to Anophelines than adults (Muirhead-Thomson, 1951; Thomas,1951). Studies on the chemical composition of human volatiles (Bernieret al., 1999; Krotoszynski et al., 1977; Labows, 1979) revealed theexistence of a large number (>350) of chemicals, and work is in progressto study the most important components of these volatiles regulatingmosquito behavior. Lastly, it is also clear that responses to CO₂ affectinter-individual differences in attractiveness (Brady et al., 1997) and,thus, CO₂ serves as a universal attractant to many mosquito species(Gillies, 1980; Takken et al., 1997; Takken and Knols, 1999). It hasbeen reported that CO₂ stimulation synergizes with host body odor andhas an activating effect on host-seeking anopheline mosquitoes, inducingtake-off and sustained flight behaviors (Dekker et al., 2001b; Gillies,1980; Mboera and Takken, 1997).

In a process that is analogous to the sense of smell in humans as wellas other insects, mosquito olfactionis initiated by the process ofchemosensory signal transduction by which chemical signals (typicallyenvironmental cues) are translated into neuronal activity and,ultimately, behavioral outputs. In An. gambiae, this takes place withinspecialized hair-like structures called sensilla that are dispersedthroughout the antennae and other head appendages on adult andlarval-stage anopheline mosquitoes (Zwiebel and Takken, 2004; FIG. 2).

Until recently, much of the inventors' view of insect olfactory signaltransduction at the molecular level has been strongly influenced byobservations made in vertebrates, crustaceans and nematodes (Hildebrandand Shepherd, 1997; Krieger and Breer, 1999). The canonical modelinvolves a family of heptahelical G-protein-coupled receptors (GPCRs)that activate downstream effectors via heterotrimeric GTP-binding (G)proteins and traditional second messengers. It has long been assumed,although not fully accepted (see below), that the canonical model ofolfactory signal transduction would also hold true in insects, in whichseveral of the “usual” molecular suspects have been identified and, inpart, functionally characterized. These include arrestins (Merrill etal., 2002; 2003; 2005), odorant-binding proteins (OBPs) (Pelosi andMaida, 1995), a heterotrimeric G-protein (Laue et al., 1997) as well asa CNG (Baumann et al., 1994; Krieger et al., 1999) and an IP3-gated ionchannel (Stengl, 1994). In one study using the cockroach, it wasdemonstrated that pheromone exposure of insect antennal preparationscaused a rapid increase in IP3 levels (Breer et al., 1990), which in afollow-up study could be inhibited by pertussis toxin (Boekhoff et al.,1990), indicating that the IP3 increase is dependent on either a Gαi ora Gαo G-protein subunit. More recently, the inventors carried out amolecular survey of G-protein expression in the olfactory appendages ofAn. gambiae, in which Gαq localization consistent with involvement inolfactory signal transduction was observed along the dendrites of mostolfactory sensory neurons (Rutzler et al., 2006). Furthermore, pheromonereceptor neuron activity of Bombyx mori could be stimulated withfluoride ions (Laue et al., 1997), which are known to activateheterotrimeric G proteins via binding to the a subunit in combinationwith magnesium ions (Antonny et al., 1993). However, despite thisgrowing wealth of information, the precise mode of insect olfactorysignal transduction remains largely obscure and is therefore the subjectof ongoing investigation that has raised serious issues with regard tothe validity of GPCR-based paradigms.

Because olfaction was mediated by GPCRs in both vertebrates and at leastone invertebrate, it was assumed that insects would also utilize theseproteins in olfactory signal transduction. Indeed, using a variety ofapproaches, a large family of candidate ORs has been identified in D.melanogaster (Clyne et al., 1999) (Gao and Chess, 1999; Vosshall et al.,1999). In the first of these studies, putative D. melanogaster Ors(Dors) were identified using a novel computer algorithm that searchedfor conserved physicochemical features common to known transmembraneproteins (Kim et al., 2000) rather than relying on a sequencehomology-based screen (which might miss a divergent member of aparticular family). The structures that were ultimately identified usingthese strategies led to the identification of a highly divergent familyof receptors, displaying between 10% and 75% identity and bearing nosignificant homology to any other GPCR family (Smith, 1999). Anotherchemosensory receptor family was also described in D. melanogaster andAn. gambiae and is presumed to comprise gustatory (taste) receptors(Clyne et al., 2000; Hill et al., 2002; Scott et al., 2001). The othercircumstantial criterion to infer olfactory function has been providedby various in situ expression pattern studies that have demonstratedthat the majority of these genes were selectively and stereotypicallyexpressed in the fly olfactory sensory neurons (Clyne et al., 1999)(Elmore and Smith, 2001; Gao and Chess, 1999; Vosshall, 2001; Vosshallet al., 1999). Two-color (double-labeling) in situ hybridizationsuggests that, with two notable caveats (Goldman et al., 2005), most D.melanogaster ORNs are likely to express a single DOR gene (Vosshall etal., 2000), which is analogous to mammalian systems (Mombaerts, 1999),but in stark contrast to the C. elegans system. One apparent exceptionto the one ORN-one receptor principle is the non-conventional DOR83b,now known as DmORco. Unlike most other DORs, DmORco is expressedthroughout the majority of antennal and maxillary palp ORNs of D.melanogaster. Putative DmORco orthologs have been identified in a widerange of insect species and share many characteristics, including highsequence identity (Pitts et al., 2004), characteristic broad expressionpattern (Krieger et al., 2003) and conserved functions (Jones et al.,2005). ORco family members are considered non-conventional ORs as theyact as general dimerization partners for other members of the DOR family(Larsson et al., 2004). More recently, Benton, Vosshall and co-workershave identified a novel set of ionotropic glutamate receptors as a newclass of insect chemosensory receptors (IRs) that are expressed inDmOrco-ORNs associated with coeloconic sensilla where they act inparallel with “classical” insect ORs to respond to ammonia and otherenvironmental cues (Benton et al., 2009; Liu et al., 2010).

Elegant studies by the Vosshall lab have also suggested that insect ORsmanifest a novel topology relative to vertebrate ORs (Benton et al.,2006). In the absence of actual structural information insect ORs havebeen structurally characterized largely based on bioinformatic modelsderived from vertebrates (Clyne et al., 2000; Vosshall et al., 1999).Indeed, while sequence-based phylogenies recognize that insect ORs ingeneral comprise a distinct family of heptahelical receptors that are anexpanded lineage of ancestral chemosensory receptors (Mombaerts, 1999;Robertson et al., 2003) there is a growing awareness that insect ORs arelikely to represent a structurally unique set of sensory proteins. Thesestudies provide compelling evidence in support of the view thatDrosophila ORs are heteromeric complexes between the non-conventionalDmORco and conventional, odorant binding DORs that adopt a novelmembrane topology in which the N-terminus is intracellular rather thanthe extra-cellular localization that is typical of vertebrate ORs andGPCRs (Benton et al., 2006). Independent validation (Lundin et al.,2007) together with recent computational analyses employing hiddenMarkov modeling that “strongly rejects” classifying arthropod ORs asGPCRs (Wistrand et al., 2006) raise significant concerns regarding thenature of the signaling pathways that are downstream of odorantactivation in insects. Indeed, two recent studies provide provocativeevidence to suggest that Drosophila ORs manifest properties of bothligand-gated (Sato et al., 2008) and cyclic-nucleotide-gated ionchannels (Wicher et al., 2008). While these hypotheses still differ intheir particulars, there is growing awareness that insect olfactorytransduction may diverge from vertebrate paradigms and act asnon-GPCR-mediated ion-channels (FIG. 2). In any case, while currenthypotheses may differ, the growing possibility that insect olfactorytransduction may diverge from vertebrate paradigms and act vianon-GPCR-mediated mechanisms such as ion channels (FIG. 2) arecompelling.

In the first report of insect ORs outside of the model insect system D.melanogaster, members of the inventors' laboratory, as part of acollaborative effort with Drs. John Carlson and Hugh Robertson, wereresponsible for the identification of a set of candidate Or genesselectively expressed in olfactory tissues of An. gambiae (AgORs) (Foxet al., 2001). Moreover, that report also demonstrated that at least oneof the initial set of AgORs displays female-specific expression, afeature that may be especially relevant for disease transmission. In asubsequent study, as part of the effort to annotate the recentlycompleted genomic sequence of An. gambiae (Holt et al., 2002), theinventors (in collaboration with other groups) utilized bioinformaticsand molecular approaches to describe the entire An. gambiae GPCR genefamily (AgGPCRs); of the 275 putative AgGPCRs, 79 candidate AgORs weredescribed (Hill et al., 2002). Furthermore, a similar bioinformaticapproach (using a non-public database) has been used to identify ninecandidate Or genes in the heliothine moth Heliothis virescens (Kriegeret al., 2002), some of which share sequence homology with AgOrs. Morerecently, a large family of candidate Or genes have been identified inthe genome sequence of the honey bee, Apis mellifera (Robertson andWanner, 2006), Ae. aegypti (Bohbot et al., 2007) and the red flourbeetle, Tribolium casteneum (Engsontia et al., 2008).

Thus far, insect ORs have been extensively deorphanized in a number ofheterologous systems. The first successful functional studies of insectORs were carried out for DOR43a using a Xenopus oocyte expression system(Wetzel et al., 2001), and over-expression in D. melanogaster (Storkuhland Kettler, 2001) showed increased sensitivity to a set of fourodorants. The Carlson laboratory has used a novel experimental approachthat takes advantage of a genetic strain of D. melanogaster in which achromosomal deletion has resulted in the loss of the endogenousreceptors (DOR22a/b) from the ab3A ORN. The resultant formation of a the“empty neuron” system facilitates the specific targeting of exogenous ORgenes into the empty neuron, thereby allowing electrophysiologicalassessment of the ability of the novel receptor to carry outchemosensory signal transduction within the ab3A neuron upon stimulationwith a diverse set of odorants (Dobritsa et al., 2003). This system hasbeen used effectively to functionally characterize nearly all the DORs(Hallem et al., 2004a) (Hallem and Carlson, 2006), leading to a highlydeveloped map of the multidimensional “odor space” of the DORs. As partof a long-standing collaboration between the Carlson lab and that of theinventors, multiple AgOR have also been functionally characterized inthe Drosophila empty neuron (Hallem et al., 2004b; Lu et al., 2007).These studies, along with the success in functionally expressing over 40AgORs in Xenopus and cell culture systems have lead to significantadvances in understanding the molecular basis for olfactory sensitivityin larval (Xia et al., 2008) and adult (Lu et al., 2007) An. gambiae.For example, CO₂ which acts as universal attractant for many species ofmosquitoes (Takken and Knols, 1999) elicits avoidance in Drosophilawhere it has been identified as an active component of the “stressodorant” that targets a discrete population of sensory neurons (Suh etal., 2007) and where a pair of highly conserved putative gustatoryreceptors (Gr21a and Gr63a) have been shown to both be both necessaryand sufficient to mediate olfactory sensitivity to CO2 in Drosophila(Jones et al., 2007; Kwon et al., 2007). As part of a comprehensivestudy of the olfactory processes on the maxillary palp in An gambiae,the inventors have identified three Gr21a/63a homologs (AgGrs22-24) asthe molecular partners required that together comprise the anophelineCO₂ receptor (Lu et al., 2007).

IV. ACTIVE AGENTS

In accordance with the present invention, there is provided an agentshown below and designated throughout as VUAA1:

This compound was identified through the screening procedure describedin the Examples.

It is also contemplated that that the concentrations of the active agentcan vary. In non-limiting embodiments, for example, the compositions mayinclude in their final form, for example, at least about 0.0001%,0.0002%, 0.0003%, 0.0004%, 0.0005%, 0.0006%, 0.0007%, 0.0008%, 0.0009%,0.0010%, 0.0011%, 0.0012%, 0.0013%, 0.0014%, 0.0015%, 0.0016%, 0.0017%,0.0018%, 0.0019%, 0.0020%, 0.0021%, 0.0022%, 0.0023%, 0.0024%, 0.0025%,0.0026%, 0.0027%, 0.0028%, 0.0029%, 0.0030%, 0.0031%, 0.0032%, 0.0033%,0.0034%, 0.0035%, 0.0036%, 0.0037%, 0.0038%, 0.0039%, 0.0040%, 0.0041%,0.0042%, 0.0043%, 0.0044%, 0.0045%, 0.0046%, 0.0047%, 0.0048%, 0.0049%,0.0050%, 0.0051%, 0.0052%, 0.0053%, 0.0054%, 0.0055%, 0.0056%, 0.0057%,0.0058%, 0.0059%, 0.0060%, 0.0061%, 0.0062%, 0.0063%, 0.0064%, 0.0065%,0.0066%, 0.0067%, 0.0068%, 0.0069%, 0.0070%, 0.0071%, 0.0072%, 0.0073%,0.0074%, 0.0075%, 0.0076%, 0.0077%, 0.0078%, 0.0079%, 0.0080%, 0.0081%,0.0082%, 0.0083%, 0.0084%, 0.0085%, 0.0086%, 0.0087%, 0.0088%, 0.0089%,0.0090%, 0.0091%, 0.0092%, 0.0093%, 0.0094%, 0.0095%, 0.0096%, 0.0097%,0.0098%, 0.0099%, 0.0100%, 0.0200%, 0.0250%, 0.0275%, 0.0300%, 0.0325%,0.0350%, 0.0375%, 0.0400%, 0.0425%, 0.0450%, 0.0475%, 0.0500%, 0.0525%,0.0550%, 0.0575%, 0.0600%, 0.0625%, 0.0650%, 0.0675%, 0.0700%, 0.0725%,0.0750%, 0.0775%, 0.0800%, 0.0825%, 0.0850%, 0.0875%, 0.0900%, 0.0925%,0.0950%, 0.0975%, 0.1000%, 0.1250%, 0.1500%, 0.1750%, 0.2000%, 0.2250%,0.2500%, 0.2750%, 0.3000%, 0.3250%, 0.3500%, 0.3750%, 0.4000%, 0.4250%,0.4500%, 0.4750%, 0.5000%, 0.5250%, 0.0550%, 0.5750%, 0.6000%, 0.6250%,0.6500%, 0.6750%, 0.7000%, 0.7250%, 0.7500%, 0.7750%, 0.8000%, 0.8250%,0.8500%, 0.8750%, 0.9000%, 0.9250%, 0.9500%, 0.9750%, 1.0%, 1.1%, 1.2%,1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%,2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%,3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%,4.9%, 5.0%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6.0%,6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7.0%, 7.1%, 7.2%,7.3%, 7.4%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, 8.0%, 8.1%, 8.2%, 8.3%, 8.4%,8.5%, 8.6%, 8.7%, 8.8%, 8.9%, 9.0%, 9.1%, 9.2%, 9.3%, 9.4%, 9.5%, 9.6%,9.7%, 9.8%, 9.9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%,21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or any range derivabletherein. In non-limiting aspects, the percentage can be calculated byweight or volume of the total composition. A person of ordinary skill inthe art would understand that the concentrations can vary depending onthe addition, substitution, and/or subtraction of the compounds, agents,or active ingredients, to the disclosed methods and compositions.

V. FORMULATIONS FOR ACTIVE AGENTS

In one embodiment, the present agent provides topical formulationsincluding agents of the present invention. Including the active agent,such formulations will contain a variety of compounds and compositionsthat are typical for use with topical delivery. The following is adiscussion of agents for use in preparation of topical formulations.

A. Film Forming Agents

Film formers are materials or compound, which, upon drying, can producea continuous film on skin. This can increase the durability of acomposition while also resulting in reduced moisture loss from skin. TheCTFA Handbook at volume 3, pages 3187-3192, provides a wide range offilm formers that can be used in the context of the present invention,all of which are incorporated by reference. Non-limiting examples ofsuch film formers include Polysilicone-6, Polysilicone-8,Polysilicone-11, Polysilicone-14,VP/Dimethiconylacrylate/Polycarbamyl/Polyglycol Ester,VP/Dimethylaminoethylmethacrylate Copolymer,VP/Dimethylaminoethylmethacrylate/Polycarbamyl Polyglycol Ester,VP/Eicosene Copolymer, VP/Hexadecene Copolymer, VP/Methacrylamide/VinylImidazole Copolymer, VP/Polycarbamyl Polyglycol Ester, VP/VA Copolymer,Polyester-1, Polyester-2, Polyester-3, Polyester-4, Polyester-5,Polyester-7, Polyester-8, and Polyester-10.

B. Ester Containing Solvents

Esters are covalent compounds formed between acids and alcohols. Theycan be used to stabilize and solubilize agents in the context of thepresent invention. The CTFA Handbook at volume 3, pages 3079-3088,provides a wide range of ester containing solvents that can be used inthe context of the present invention, all of which are incorporated byreference. Non-limiting examples of such solvents include C12-15 Alkylbenzoate, neopentyl glycol diheptanoate, dipropylene glycol dibenzoate,and PPG-15 stearyl ether benzoate.

C. Gelling Agents

The composition of the present invention can be formulated as atransparent gel. Gelling agents such as dimethicone/bis-isobutyl PPG-20crosspolymer can used to create the gel-based primer. Further, a widerange of gelling agents are commercially available from Dow Corning(Midland, Mich. (USA)). A non-limiting example includes Dow CorningEL-8050 ID, which is a blend of dimethicone/bis-isobutyl PPG-20crosspolymer and isododecane.

D. Additional Skin Conditioning Agents and Emollients

Non-limiting examples of skin conditioning agents and emollients thatcan be used with the compositions of the present invention include aminoacids, chondroitin sulfate, diglycerin, erythritol, fructose, glucose,glycerol polymers, glycol, 1,2,6-hexanetriol, honey, hyaluronic acid,hydrogenated honey, hydrogenated starch hydrolysate, inositol, lactitol,maltitol, maltose, mannitol, natural moisturizing factor, PEG-15butanediol, polyglyceryl sorbitol, salts of pyrollidone carboxylic acid,potassium PCA, propylene glycol, sodium glucuronate, sodium PCA,sorbitol, sucrose, trehalose, urea, and xylitol.

Other examples include acetylated lanolin, acetylated lanolin alcohol,acrylates/C10-30 alkyl acrylate crosspolymer, acrylates copolymer,alanine, algae extract, aloe barbadensis, aloe-barbadensis extract, aloebarbadensis gel, althea officinalis extract, aluminum starchoctenylsuccinate, aluminum stearate, apricot (prunus armeniaca) kerneloil, arginine, arginine aspartate, arnica montana extract, ascorbicacid, ascorbyl palmitate, aspartic acid, avocado (persea gratissima)oil, barium sulfate, barrier sphingolipids, butyl alcohol, beeswax,behenyl alcohol, beta-sitosterol, BHT, birch (betula alba) bark extract,borage (borago officinalis) extract, 2-bromo-2-nitropropane-1,3-diol,butcherbroom (ruscus aculeatus) extract, butylene glycol, calendulaofficinalis extract, calendula officinalis oil, candelilla (euphorbiacerifera) wax, canola oil, caprylic/capric triglyceride, cardamon(elettaria cardamomum) oil, carnauba (copernicia cerifera) wax,carrageenan (chondrus crispus), carrot (daucus carota sativa) oil,castor (ricinus communis) oil, ceramides, ceresin, ceteareth-5,ceteareth-12, ceteareth-20, cetearyl octanoate, ceteth-20, ceteth-24,cetyl acetate, cetyl octanoate, cetyl palmitate, chamomile (anthemisnobilis) oil, cholesterol, cholesterol esters, cholesterylhydroxystearate, citric acid, clary (salvia sclarea) oil, cocoa(theobroma cacao) butter, coco-caprylate/caprate, coconut (cocosnucifera) oil, collagen, collagen amino acids, corn (zea mays) oil,fatty acids, decyl oleate, dextrin, diazolidinyl urea, dimethiconecopolyol, dimethiconol, dioctyl adipate, dioctyl succinate,dipentaerythrityl hexacaprylate/hexacaprate, DMDM hydantoin, DNA,erythritol, ethoxydiglycol, ethyl linoleate, eucalyptus globulus oil,evening primrose (oenothera biennis) oil, fatty acids, tructose,gelatin, geranium maculatum oil, glucosamine, glucose glutamate,glutamic acid, glycereth-26, glycerol, glyceryl distearate, glycerylhydroxystearate, glyceryl laurate, glyceryl linoleate, glycerylmyristate, glyceryl oleate, glyceryl stearate, glyceryl stearate SE,glycine, glycol stearate, glycol stearate SE, glycosaminoglycans, grape(vitis vinifera) seed oil, hazel (corylus americana) nut oil, hazel(corylus avellana) nut oil, hexylene glycol, honey, hyaluronic acid,hybrid safflower (carthamus tinctorius) oil, hydrogenated castor oil,hydrogenated coco-glycerides, hydrogenated coconut oil, hydrogenatedlanolin, hydrogenated lecithin, hydrogenated palm glyceride,hydrogenated palm kernel oil, hydrogenated soybean oil, hydrogenatedtallow glyceride, hydrogenated vegetable oil, hydrolyzed collagen,hydrolyzed elastin, hydrolyzed glycosaminoglycans, hydrolyzed keratin,hydrolyzed soy protein, hydroxylated lanolin, hydroxyproline,imidazolidinyl urea, iodopropynyl butylcarbamate, isocetyl stearate,isocetyl stearoyl stearate, isodecyl oleate, isopropyl isostearate,isopropyl lanolate, isopropyl myristate, isopropyl palmitate, isopropylstearate, isostearamide DEA, isostearic acid, isostearyl lactate,isostearyl neopentanoate, jasmine (jasminum officinale) oil, jojoba(buxus chinensis) oil, kelp, kukui (aleurites moluccana) nut oil,lactamide MEA, laneth-16, laneth-10 acetate, lanolin, lanolin acid,lanolin alcohol, lanolin oil, lanolin wax, lavender (lavandulaangustifolia) oil, lecithin, lemon (citrus medica limonum) oil, linoleicacid, linolenic acid, macadamia ternifolia nut oil, magnesium stearate,magnesium sulfate, maltitol, matricaria (chamomilla recutita) oil,methyl glucose sesquistearate, methylsilanol PCA, microcrystalline wax,mineral oil, mink oil, mortierella oil, myristyl lactate, myristylmyristate, myristyl propionate, neopentyl glycol dicaprylate/dicaprate,octyldodecanol, octyldodecyl myristate, octyldodecyl stearoyl stearate,octyl hydroxystearate, octyl palmitate, octyl salicylate, octylstearate, oleic acid, olive (olea europaea) oil, orange (citrusaurantium dulcis) oil, palm (elaeis guineensis) oil, palmitic acid,pantethine, panthenol, panthenyl ethyl ether, paraffin, PCA, peach(prunus persica) kernel oil, peanut (arachis hypogaea) oil, PEG-8 C12-18ester, PEG-15 cocamine, PEG-150 distearate, PEG-60 glyceryl isostearate,PEG-5 glyceryl stearate, PEG-30 glyceryl stearate, PEG-7 hydrogenatedcastor oil, PEG-40 hydrogenated castor oil, PEG-60 hydrogenated castoroil, PEG-20 methyl glucose sesquistearate, PEG40 sorbitan peroleate,PEG-5 soy sterol, PEG-10 soy sterol, PEG-2 stearate, PEG-8 stearate,PEG-20 stearate, PEG-32 stearate, PEG40 stearate, PEG-50 stearate,PEG-100 stearate, PEG-150 stearate, pentadecalactone, peppermint (menthapiperita) oil, petrolatum, phospholipids, polyamino sugar condensate,polyglyceryl-3 diisostearate, polyquaternium-24, polysorbate 20,polysorbate 40, polysorbate 60, polysorbate 80, polysorbate 85,potassium myristate, potassium palmitate, potassium sorbate, potassiumstearate, propylene glycol, propylene glycol dicaprylate/dicaprate,propylene glycol dioctanoate, propylene glycol dipelargonate, propyleneglycol laurate, propylene glycol stearate, propylene glycol stearate SE,PVP, pyridoxine dipalmitate, quaternium-15, quaternium-18 hectorite,quaternium-22, retinol, retinol palmitate, rice (oryza sativa) bran oil,RNA, rosemary (rosmarinus officinalis) oil, rose oil, safflower(carthamus tinctorius) oil, sage (salvia officinalis) oil, salicylicacid, sandalwood (santalum album) oil, serine, serum protein, sesame(sesamum indicum) oil, silk powder, sodium chondroitin sulfate, sodiumhyaluronate, sodium lactate, sodium palmitate, sodium PCA, sodiumpolyglutamate, sodium stearate, soluble collagen, sorbic acid, sorbitanlaurate, sorbitan oleate, sorbitan palmitate, sorbitan sesquioleate,sorbitan stearate, sorbitol, soybean (glycine soja) oil, sphingolipids,squalane, squalene, stearamide MEA-stearate, stearic acid, stearoxydimethicone, stearoxytrimethylsilane, stearyl alcohol, stearylglycyrrhetinate, stearyl heptanoate, stearyl stearate, sunflower(helianthus annuus) seed oil, sweet almond (prunus amygdalus dulcis)oil, synthetic beeswax, tocopheryl linoleate, tridecyl neopentanoate,tridecyl stearate, triethanolamine, tristearin, urea, vegetable oil,water, waxes, wheat (triticum vulgare) germ oil, and ylang ylang(cananga odorata) oil.

E. Antioxidants

Non-limiting examples of antioxidants that can be used with thecompositions of the present invention include acetyl cysteine, ascorbicacid polypeptide, ascorbyl dipalmitate, ascorbyl methylsilanolpectinate, ascorbyl palmitate, ascorbyl stearate, BHA, BHT, t-butylhydroquinone, cysteine, cysteine HCl, diamylhydroquinone,di-t-butylhydroquinone, dicetyl thiodipropionate, dioleyl tocopherylmethylsilanol, disodium ascorbyl sulfate, distearyl thiodipropionate,ditridecyl thiodipropionate, dodecyl gallate, erythorbic acid, esters ofascorbic acid, ethyl ferulate, ferulic acid, gallic acid esters,hydroquinone, isooctyl thioglycolate, kojic acid, magnesium ascorbate,magnesium ascorbyl phosphate, methylsilanol ascorbate, natural botanicalanti-oxidants such as green tea or grape seed extracts,nordihydroguaiaretic acid, octyl gallate, phenylthioglycolic acid,potassium ascorbyl tocopheryl phosphate, potassium sulfite, propylgallate, quinones, rosmarinic acid, sodium ascorbate, sodium bisulfite,sodium erythorbate, sodium metabisulfite, sodium sulfite, superoxidedismutase, sodium thioglycolate, sorbityl furfural, thiodiglycol,thiodiglycolamide, thiodiglycolic acid, thioglycolic acid, thiolacticacid, thiosalicylic acid, tocophereth-5, tocophereth-10, tocophereth-12,tocophereth-18, tocophereth-50, tocophersolan, tocopheryl linoleate,tocopheryl nicotinate, tocopheryl succinate, andtris(nonylphenyl)phosphite.

F. Structuring Agents

In other non-limiting aspects, the compositions of the present inventioncan include a structuring agent. Structuring agents, in certain aspects,assist in providing rheological characteristics to the composition tocontribute to the composition's stability. In other aspects, structuringagents can also function as an emulsifier or surfactant. Non-limitingexamples of structuring agents include stearic acid, palmitic acid,stearyl alcohol, cetyl alcohol, behenyl alcohol, stearic acid, palmiticacid, the polyethylene glycol ether of stearyl alcohol having an averageof about 1 to about 21 ethylene oxide units, the polyethylene glycolether of cetyl alcohol having an average of about 1 to about 5 ethyleneoxide units, and mixtures thereof.

G. Emulsifiers

In some non-limiting aspects, the compositions can include one or moreemulsifiers. Emulsifiers can reduce the interfacial tension betweenphases and improve the formulation and stability of an emulsion. Theemulsifiers can be nonionic, cationic, anionic, and zwitterionicemulsifiers (See McCutcheon's (1986); U.S. Pat. Nos. 5,011,681;4,421,769; 3,755,560). Non-limiting examples include esters of glycerin,esters of propylene glycol, fatty acid esters of polyethylene glycol,fatty acid esters of polypropylene glycol, esters of sorbitol, esters ofsorbitan anhydrides, carboxylic acid copolymers, esters and ethers ofglucose, ethoxylated ethers, ethoxylated alcohols, alkyl phosphates,polyoxyethylene fatty ether phosphates, fatty acid amides, acyllactylates, soaps, TEA stearate, DEA oleth-3 phosphate, polyethyleneglycol 20 sorbitan monolaurate (polysorbate 20), polyethylene glycol 5soya sterol, steareth-2, steareth-20, steareth-21, ceteareth-20, PPG-2methyl glucose ether distearate, ceteth-10, polysorbate 80, cetylphosphate, potassium cetyl phosphate, diethanolamine cetyl phosphate,polysorbate 60, glyceryl stearate, PEG-100 stearate, and mixturesthereof.

H. Silicone Containing Compounds

In non-limiting aspects, silicone containing compounds include anymember of a family of polymeric products whose molecular backbone ismade up of alternating silicon and oxygen atoms with side groupsattached to the silicon atoms. By varying the —Si—O-chain lengths, sidegroups, and crosslinking, silicones can be synthesized into a widevariety of materials. They can vary in consistency from liquid to gel tosolids.

The silicone containing compounds that can be used in the context of thepresent invention include those described in this specification or thoseknown to a person of ordinary skill in the art. Non-limiting examplesinclude silicone oils (e.g., volatile and non-volatile oils), gels, andsolids. In preferred aspects, the silicon containing compounds includesa silicone oils such as a polyorganosiloxane. Non-limiting examples ofpolyorganosiloxanes include dimethicone, cyclomethicone, phenyltrimethicone, trimethylsilylamodimethicone, stearoxytrimethylsilane, ormixtures of these and other organosiloxane materials in any given ratioin order to achieve the desired consistency and applicationcharacteristics depending upon the intended application (e.g., to aparticular area such as the skin, hair, or eyes). A “volatile siliconeoil” includes a silicone oil have a low heat of vaporization, i.e.normally less than about 50 cal per gram of silicone oil. Non-limitingexamples of volatile silicone oils include: cyclomethicones such as DowCorning 344 Fluid, Dow Corning 345 Fluid, Dow Corning 244 Fluid, and DowCorning 245 Fluid, Volatile Silicon 7207 (Union Carbide Corp., Danbury,Conn.); low viscosity dimethicones, i.e., dimethicones having aviscosity of about 50 cst or less (e.g., dimethicones such as DowCorning 200-0.5 cst Fluid). The Dow Corning Fluids are available fromDow Corning Corporation, Midland, Mich. Cyclomethicone and dimethiconeare described in the Third Edition of the CTFA Cosmetic IngredientDictionary (incorporated by reference) as cyclic dimethyl polysiloxanecompounds and a mixture of fully methylated linear siloxane polymersend-blocked with trimethylsiloxy units, respectively. Other non-limitingvolatile silicone oils that can be used in the context of the presentinvention include those available from General Electric Co., SiliconeProducts Div., Waterford, N.Y. and SWS Silicones Div. of StaufferChemical Co., Adrian, Mich.

I. Essential Oils

Essential oils include oils derived from herbs, flowers, trees, andother plants. Such oils are typically present as tiny droplets betweenthe plant's cells, and can be extracted by several method known to thoseof skill in the art (e.g., steam distilled, enfleurage (i.e., extractionby using fat), maceration, solvent extraction, or mechanical pressing).When these types of oils are exposed to air they tend to evaporate(i.e., a volatile oil). As a result, many essential oils are colorless,but with age they can oxidize and become darker. Essential oils areinsoluble in water and are soluble in alcohol, ether, fixed oils(vegetal), and other organic solvents. Typical physical characteristicsfound in essential oils include boiling points that vary from about 160°to 240° C. and densities ranging from about 0.759 to about 1.096.

Essential oils typically are named by the plant from which the oil isfound. For example, rose oil or peppermint oil are derived from rose orpeppermint plants, respectively. Non-limiting examples of essential oilsthat can be used in the context of the present invention include sesameoil, macadamia nut oil, tea tree oil, evening primrose oil, Spanish sageoil, Spanish rosemary oil, coriander oil, thyme oil, pimento berriesoil, rose oil, anise oil, balsam oil, bergamot oil, rosewood oil, cedaroil, chamomile oil, sage oil, clary sage oil, clove oil, cypress oil,eucalyptus oil, fennel oil, sea fennel oil, frankincense oil, geraniumoil, ginger oil, grapefruit oil, jasmine oil, juniper oil, lavender oil,lemon oil, lemongrass oil, lime oil, mandarin oil, marjoram oil, myrrhoil, neroli oil, orange oil, patchouli oil, pepper oil, black pepperoil, petitgrain oil, pine oil, rose otto oil, rosemary oil, sandalwoodoil, spearmint oil, spikenard oil, vetiver oil, wintergreen oil, orylang ylang. Other essential oils known to those of skill in the art arealso contemplated as being useful within the context of the presentinvention.

J. Thickening Agents

Thickening agents include substances that can increase the viscosity ofa composition. Thickeners include those that can increase the viscosityof a composition without substantially modifying the efficacy of theactive ingredient within the composition. Thickeners can also increasethe stability of the compositions of the present invention.

Non-limiting examples of additional thickening agents that can be usedin the context of the present invention include carboxylic acidpolymers, crosslinked polyacrylate polymers, polyacrylamide polymers,polysaccharides, and gums. Examples of carboxylic acid polymers includecrosslinked compounds containing one or more monomers derived fromacrylic acid, substituted acrylic acids, and salts and esters of theseacrylic acids and the substituted acrylic acids, wherein thecrosslinking agent contains two or more carbon-carbon double bonds andis derived from a polyhydric alcohol (see U.S. Pat. Nos. 5,087,445;4,509,949; 2,798,053). Examples of commercially available carboxylicacid polymers include carbomers, which are homopolymers of acrylic acidcrosslinked with allyl ethers of sucrose or pentaerytritol (e.g.,Carbopol™ 900 series from B. F. Goodrich).

Non-limiting examples of crosslinked polyacrylate polymers includecationic and nonionic polymers. Examples are described in U.S. Pat. Nos.5,100,660; 4,849,484; 4,835,206; 4,628,078; and 4,599,379).

Non-limiting examples of polyacrylamide polymers (including nonionicpolyacrylamide polymers including substituted branched or unbranchedpolymers) include polyacrylamide, isoparaffin and laureth-7, multi-blockcopolymers of acrylamides and substituted acrylamides with acrylic acidsand substituted acrylic acids.

Non-limiting examples of polysaccharides include cellulose,carboxymethyl hydroxyethylcellulose, cellulose acetate propionatecarboxylate, hydroxyethylcellulose, hydroxyethyl ethylcellulose,hydroxypropylcellulose, hydroxypropyl methylcellulose, methylhydroxyethylcellulose, microcrystalline cellulose, sodium cellulosesulfate, and mixtures thereof. Another example is an alkyl substitutedcellulose where the hydroxy groups of the cellulose polymer ishydroxyalkylated (preferably hydroxy ethylated or hydroxypropylated) toform a hydroxyalkylated cellulose which is then further modified with aC₁₀-C₃₀ straight chain or branched chain alkyl group through an etherlinkage. Typically these polymers are ethers of C₁₀-C₃₀ straight orbranched chain alcohols with hydroxyalkylcelluloses. Other usefulpolysaccharides include scleroglucans comprising a linear chain of (1-3)linked glucose units with a (1-6) linked glucose every three unit.

Non-limiting examples of gums that can be used with the presentinvention include acacia, agar, algin, alginic acid, ammonium alginate,amylopectin, calcium alginate, calcium carrageenan, carnitine,carrageenan, dextrin, gelatin, gellan gum, guar gum, guarhydroxypropyltrimonium chloride, hectorite, hyaluroinic acid, hydratedsilica, hydroxypropyl chitosan, hydroxypropyl guar, karaya gum, kelp,locust bean gum, natto gum, potassium alginate, potassium carrageenan,propylene glycol alginate, sclerotium gum, sodium carboyxmethyl dextran,sodium carrageenan, tragacanth gum, xanthan gum, and mixtures thereof.

K. Vehicles

The compositions of the present invention can be incorporated into alltypes of are effective in all types of vehicles. Non-limiting examplesof suitable vehicles include emulsions (e.g., water-in-oil,water-in-oil-in-water, oil-in-water, -oil-in-water-in-oil,oil-in-water-in-silicone emulsions), creams, lotions, solutions (bothaqueous and hydro-alcoholic), anhydrous bases (such as lipsticks andpowders), gels, and ointments or by other method or any combination ofthe forgoing as would be known to one of ordinary skill in the art(Remington's, 1990). Variations and other appropriate vehicles will beapparent to the skilled artisan and are appropriate for use in thepresent invention. In certain aspects, it is important that theconcentrations and combinations of the compounds, ingredients, andactive agents be selected in such a way that the combinations arechemically compatible and do not form complexes which precipitate fromthe finished product.

VI. ARTICLES OF MANUFACTURE

The present invention contemplates the use of the VUAA1 agent in themanufacture of certain items. The material may be pre-made and thendipped, painted or sprayed with the agent. Alternatively, the materialsmay be formed in the presence of the agent so as to incorporate theagent integrally thereinto.

For example, VUAA1 may be used to coat or impregnate various articles ofmanufacture, the use of which can help deliver VUAA1 to a mosquitoenvironment and/or protect a user of the article from mosquito contact.Such articles include netting, such as the type use to exclude insectsfrom dwelling (i.e., in windows and door ways) or to exclude insectsfrom a particular location, such as a bed or room.

Other articles of manufacture include clothing or fabric from whichclothing can be produced. Clothing includes hats, veils, masks, shoesand gloves, as well as shirts, pants and underwear. Other articlesinclude bedding, such as sheets, blankets, pillow cases, and mattresses.Still additional articles include tarps, tents, awnings, door flaps,screens, or drapes.

VII. AGENT DELIVERY SYSTEMS

A. Misting Systems

The active agent of the present invention may, in one embodiment, beadvantageously dispersed into an environment using a misting system. Theenvironment may be a single family dwelling yard, and street, aneighborhood, a subdivision, a township or a city. Examples of mistingsystems are shown in U.S. Pat. Nos. 7,306,167 and 7,090,147, and U.S.Patent Publication 2006/0260183, both of which are hereby incorporatedby reference.

B. Baits and Pellets

In many cases, it would be desirable to apply the agent of the presentinvention in solid form. Solid pest control compositions typically areless prone to volatile dissemination of the active agent, and in someinstances may be more readily and conveniently applied; for example,solid pest control compositions may be dropped from a helicopter orairplane or other elevated conveyance onto the surface of a large bodyof water somewhat more readily than can liquids. In addition, solidcontrol agents are believed to be more able to penetrate a vegetativecanopy when disseminated from an elevated conveyance.

When it is desired to form a solid composition for mosquitoes, a numberof criteria are desirable. First, the solid pest control compositionshould be sufficiently durable to allow the control composition to betransported in bulk, such as by rail car or via bagged transport.Second, the solid composition, which generally will include a carrierand an active control agent, must be compatible with the pest targetarea environment; consequently, the carrier should be readilybiodegradable. Third, the solid pest control composition should readilyand quickly release the control agent when applied into a water columnor when otherwise contacted by water, such as rain.

The prior art has provided numerous pest control compositions. Forinstance, U.S. Pat. No. 6,391,328 describes a process for treatingorganisms with a composition that includes a carrier, an activeingredient, and a coating. The carrier material is said to includesilica, cellulose, metal oxides, clays, paper, infusorial earth, slag,hydrophobic materials, polymers such as polyvinyl alcohol and the like.Control of the release of rate of the active ingredient is said to beobtained via choice of coating material, which is said to be a fattyacid, alcohol or ester. Similar technology purportedly is disclosed inU.S. Pat. Nos. 6,387,386; 6,350,461; 6,346,262; 6,337,078; 6,335,027;6,001,382; 5,902,596; 5,885,605; 5,858,386; 5,858,384; 5,846,553 and5,698,210 (all by Levy to Lee County Mosquito Control District, FortMeyers, Fla.).

Another pest control composition is disclosed in U.S. Pat. Nos.5,824,328, 5,567,430, 5,983,390, and 4,418,534. In accordance with thepurported teaching of these patents, the activation is provided in theform of a material that includes a super absorbent polymer and inertdiluents.

U.S. Patent Publication 2007/0160637 discloses a pest control agentformed by providing a porous starch and an active control agent absorbedwithin the porous starch, and compressing the porous starch in thepresence of heat to form discrete plural particles, including one ormore binders, and one or more secondary absorbents/fillers. The can beprepared via pelletizing in a commercial pellet mill. The particles aresufficiently durable to withstand bulk transport, such as by rail car orbag shipment, and will release the control agent quickly upon contactwith water, such that, for instance, the control agent may be releasedwhen the pest control agent is introduced to standing water.

VIII. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1: Materials & Methods

Cell Culture and Ca²⁺ Imaging.

For transient transfections, ORs were cloned into pCI (Promega) andtransfected into Flp-In™ T-REx™293 cell lines (Invitrogen) with Fu GENE6(Roche). For the creation of stable cell lines, a cell cultureexpression vector capable of expressing AgORco in conjunction with aconventional OR, pcDNA5/FRT/TO (Invitrogen) was modified to create twoindividual expression cassettes each under the control of a separateCMV/TetO₂ promoter and a BGH poly-adenylation signal. Cells (as above)were transfected with the modified pcDNA5 plasmid along with POG44 (aFlp recombinase expression plasmid) to facilitate site-specificrecombination. Stable cell lines were selected using Hygromycin B(Invitrogen). Cells were maintained in DMEM (Invitrogen) supplementedwith 10% Tetracycline-free FBS (HyClone) and 15 μg/ml Blasticidin. Forfluorometric Ca²⁺ measurements, stable lines expressing ORs of interestwere seeded at 20,000 cells/well in black wall, poly-lysine coated384-well cell culture plates (Greiner) and treated with 0.3 μg/μltetracycline (Sigma) overnight to induce OR expression. Cells weredye-loaded with 1.8 μM Fluo-4 AM (Molecular Probes), 2.5 mM Probenecid(Molecular Probes) in assay buffer (20 mM HEPES, 1×HBSS) for 45 minutesat 37° C. in 5% CO₂ prior to each assay. Ca²⁺ mobilization was assayedin an FDSS6000 (Hamamatsu). Baseline readings were taken for 20s beforeautomated addition of compound previously diluted in DMSO and assaybuffer. Ratios were described as Maximum/Minimum response and eachresponse was normalized to the maximum responder.

Chemicals.

VUAA1 was purchased from Sigma-Aldrich's Rare Chemical Library (CAS#525582-84-7). At time of print, VUAA1 was no longer available fromSigma-Aldrich.

To ensure that observed activity was elicited from VUAA1, and not from acontaminant present in the mixture, the inventors performed preparativeHigh Performance Liquid Chromatography (HPLC). Briefly, 20 mg of VUAA1was dissolved in a 50/50 mixture of methanol and DMSO and HPLC wasperformed on a Phenomenex Luna 30×50 mm C18 prep column with 0.1%Trifluoracetic acid (TFA) in H₂O coupled to an acetonitrile gradient.Appropriate fractions were pooled and passed over a TFA scavenger column(Polymer labs, StratoSpheres SPE PL-HCO3 MP-resin). The solvent wasremoved by rotary evaporation with a Biotage V10 Roto-vap, yieldingwhite powder. VUAA1 was subsequently re-dissolved in DMSO and assayed asdescribed.

Characterization of Chemical Materials.

¹H-NMR (400 MHz, DMSO-_(d6)) d 8.73 (d, J=1.8 Hz, 1H), 8.65 (dd, J=1.5,4.8 Hz, 1H), 7.97 (dt, J=1.9, 8.0 Hz, 1H), 7.49 (dd, J=2.5, 8.3 Hz, 1H),7.37 (d, J=8.4 Hz, 2H), 7.04 (d, J=8.4 HZ, 2H), 4.10 (s, 1H), 3.95 (q,J=7.2 Hz, 2H), 2.43 (q, J=7.6 Hz, 2H), 1.13 (t, J=8.0 Hz, 3H), 1.04 (t,J=8.0 Hz, 3H). ¹³C-NMR (400 MHz, DMSO-_(d6)) d 165.71, 152.92, 151.32,150.95, 149.07, 139.35, 136.87, 136.33, 128.38, 124.34, 123.90, 119.58,37.91, 27.97, 16.05, 15.42. HRMS (m/z) [M]⁺ calculated for C₁₉H₂₂N₅OS,368.1544 found 368.1545.

Patch-Clamp Recording in HEK Cells.

Currents from OR-expressing HEK293 cells were amplified with an Axopatch200b Amplifier (Axon Instruments) and digitized through a Digidata 1322A(Axon Instruments). Electrophysiological data was recorded and analyzedusing pCLAMP 10 (Axon Instruments). Electrodes were fabricated fromquartz tubing (Sutter Instruments) and pulled to 4-6 MΩ for whole cellrecording. Electrodes were filled with internal solution (120 mM KCl, 30mM D-glucose, 10 mM HEPES, 2 mM MgCl₂, 1.1 mM EGTA, and 0.1 CaCl₂ (pH7.35, 280 mOsm). External (bath) solution contained 130 mM NaCl, 34 mMD-glucose, 10 mM HEPES, 1.5 mM CaCl₂, 1.3 mM KH₂PO₄, and 0.5 MgSO₄ (pH7.35, 300 mOsm). Compounds were diluted in external solution and locallyperfused to the recording cell using Perfusion Pencil (AutomateScientific) and controlled by a ValveLink 8.2 controller (AutomateScientific). Whole cell recordings were sampled at 10 kHz and filteredat 5 kHz. Outside-out patches were obtained using 10-15MΩ electrodespulled from standard glass capillaries (World Precision Instruments) andfire-polished with an MF-830 micro forge (Narishige). Single channelrecordings were sampled at 20 kHz. Recordings were reduced to 1 kHz andlow-pass filtered at 500 Hz for display and analysis using QuB (SUNY atBuffalo).

Single Sensillum Recordings.

Single sensillum recordings were performed on 4-7 day old, non-bloodfedAnopheles gambiae females maintained on 10% sucrose and a 12/12 lightdark cycle. Legs, wings and antennae were removed from cold-anesthetizedfemales that were then restrained on double-stick tape with thread. Aglass reference electrode filled with Sensillar lymph ringers (SLR)(Xu,2005) was placed in the eye and the recording electrode filled with DMSOor VUAA1 diluted in SLR was used to puncture sensilla at their base.Responses were recorded and digitized using a Syntech IDAC-4 andanalyzed with AutoSpike software (Syntech). New glass recording pipetteswere used for every recording. Data was sampled at 12 kHz.

Example 2—Results

As part of an ongoing, cell based calcium imaging screen for novelsmall-molecule modulators of AgORs that might disrupt olfactory-drivenmosquito behaviors (Rinker et al manuscript in preparation), theinventors identified a number of compounds that activatedAgOR10+AgORco-expressing human embryonic kidney (HEK293) cells. One ofthese compounds (FIG. 3A), denoted here as VUAA1, elicited activityconsistent with allosteric agonism and was pursued for its novelproperties. The identity of VUAA1 was verified using high-resolutionmass spectrometry (HRMS) as well as ¹H and ¹³C NMR. When AgORco+AgOR10cells were tested in a plate-based calcium imaging system, VUAA1elicited concentration-dependent responses that were not seen in controlcells (FIG. 3B). Upon further investigation, VUAA1 proved capable ofactivating other AgORco7+AgOR10 cell lines as well (unpublished data).As AgORco was the common element among these functional responses, theinventors postulated that VUAA1 was a potential AgORco agonist.

To test the hypothesis that VUAA1 directly agonized AgORco, whole-cellpatch clamp responses were examined in AgORco+AgOR10 expressing cells aswell HEK293 cells stably expressing AgORco alone. In these experiments,VUAA1 elicited concentration-dependent inward currents in bothAgORco+AgOR10 and AgORco expressing cells (FIGS. 3D-E). TheVUAA1-dependent currents in AgORco+AgOR10 cells resembled thoseresulting from application of benzaldehyde, an orthosteric agonist ofAgOR10 (FIG. 3C) (Wang, 2010; Carey, 2010). AgORco+AgOR10 cells weremore sensitive to VUAA1 than AgORco cells, producing inward currents at−5.0 log M, a concentration at which AgORco had no response. Allcurrents induced by VUAA1 were AgORco-dependent; no responses wereobserved in control cells. To investigate the specificity of VUAA1agonism, the inventors transiently transfected HEK cells with the AgORcoorthologs of Drosophila melanogaster and Heliothis virescens, DmOR83band HvOR2 respectively. In cells expressing either ortholog, VUAA1elicited robust inward currents similar to AgORco-expressing cells (FIG.3F). These results demonstrate that VUAA1 is a broad-spectrum 83b familyagonist, capable of activating non-conventional ORs within and acrossmultiple insect orders. This activity is consistent with their highsequence identities (76% to DmOR83b and 67% to HvOR2) and demonstratedfunctional overlap (Jones et al., 2005).

To further investigate the conductive properties of AgORco7, theinventors determined the current-voltage relationships of AgORco+AgOR10complexes as well as AgORco on its own. Currents induced by VUAA-1 orbenzaldehyde in AgORco+AgOR10-expressing cells, and those induced byVUAA-1 in AGORco cells, were all nearly symmetrical (FIGS. 4A-D andFIGS. 5A-D). The reversal potentials of AgORco+AgOR10 complexes were−2.9±1.4 mV (benzaldehyde) and −4.8±3.0 mV (VUAA1) while AgORco alone,in the presence of VUAA1 was +0.4±1.1 mV (mean±s.e.m. FIGS. 4A-C). Thesecurrent-voltage relationships do not indicate any voltage-dependentgating, and the near-zero reversal potentials are consistent withprevious reports of insect OR complexes that suggested non-selectivecation conductance (Sato et al., 2008; Wicher et al., 2008). Theinventors next examined whether VUAA1 responses could be attenuated byruthenium red (RR), a promiscuous cation channel blocker previouslyfound to block insect OR currents. Application of RR reduced thebenzaldehyde and VUAA1-elicited currents of AgOrco+AgOR10 cells by87.8±1.8% and 68.3±2.8%, respectively (FIGS. 5A-D) while RR reducedVUAA1 responses of AgORco cells by 79.4±4.0% (FIGS. 5C-D). In additionto demonstrating that AgORco+AgOR10 complexes, and AgORco alone act asfunctional, ligand-gated ion channels, these studies also show thatVUAA1 elicits AgOR currents similar to those in response to odorants. Todetermine the broad-spectrum specificity of VUAA1, the inventors testedVUAA1 on another non-selective cation channel, transient receptorpotential vanilloid receptor 1 (TRPV1) (Caterina, 1997; Bohlen, 2010).Capsaicin, but not VUAA1 elicited a robust response in these HEK cells(FIGS. 7A-F). These results demonstrate that VUAA1 is specific to 83borthologs, and that VUAA1 is not a broad-spectrum activation of allcation channels

The inventors next examined whether activation of AgORco involves secondmessenger-based signaling, which has been reported to contribute toinsect olfactory signaling (Wicher, 2008). In these studies, which areconsistent with a previously published report (Sato et al., 2008), twocyclic nucleotide analogs (8-Br cAMP and 8-Br cGMP) were unable to evokewhole-cell currents in AgORco or AgORco+AgOR10 cells, while in bothinstances OR function was validated by subsequent application of VUAA1and benzaldehyde, respectively (FIGS. 8A-D). While the precise mechanismof signal transfer between a conventional OR and AgORco remains unknown,it is important to note that all channel properties are consistentbetween and within AgORco and AgOrco+AgOR10 complexes. Taken together,the data suggest that the channel properties of AgORco are notsignificantly altered when complexed with other AgORs and that theionotropic conductance of AgORco is the principal signaling component offunctional AgOR complexes.

In the next set of studies, outside-out membrane patches were excisedfrom AgORco-expressing cells to examine single-channel currents evokedby VUAA1 (FIG. 5A). Here, spontaneous channel opening was observedbefore VUAA1 stimulation, but with very low probability (P_(o)=0.02)(FIG. 5B). During a 5s application of VUAA1 channel opening probabilityincreased to P_(o)=0.38 (FIG. 5C). Subsequent to agonist washout,channel opening probability decreased to 0.00 (FIG. 5D). The averageunitary current of AgORco was 1.3±0.3 pA (mean±st. dev.) (FIG. 5C,inset) which is consistent with earlier single-channel studies of insectORs (Sato et al., 2008). Taken together, these data support ourhypothesis that VUAA1 can agonize AgORco in the absence of otherintracellular components and provide additional support for the role ofVUAA1 as a direct agonist of AgORco and other OR83b family members.

The inventors next performed single unit, extracellularelectrophysiological recordings on adult female An. gambiae to determinewhether VUAA1 could activate AgORco-expressing odorant receptor neurons(ORNs) in vivo. ORNs, which express AgORco and a conventional OR areenclosed within the hair-like sensilla present on olfactory tissues. Thehighly stereotypic capitate peg (Cp) sensilla, which are found on themaxillary palp, contain two ORco expressing neurons (CpB and CpC) aswell as a CO₂ sensitive neuron (CpA), which does not express AgORco (Luet al., 2007). CpA is clearly distinguished from CpB/C by its largeaction potential amplitude. The action potential amplitudes of CpB andCpC are much smaller and in some preparations indistinguishable fromeach other; as a result, the spike activity of CpB and CpC neurons werebinned for data analysis. Accordingly, the inventors would expect thatif VUAA1 is a specific AgORco agonist, it should selectively increasethe spike frequency of the CpB and CpC neurons but have no effect on CpAresponses.

Due to its relatively high molecular weight, volatile delivery of VUAA1was not feasible. As a result, VUAA1 was directly added to eachsensillum via the glass-recording electrode where VUAA1 increased thespike frequency of CpB/C neurons in a dose-dependent manner; vehiclealone had no effect (FIGS. 6A, B, D). Differential CpB/C spike activitywas observed immediately after puncturing each sensillum, suggestingmillisecond compound diffusion rates into the sensillum (FIGS. 6A, B,D). At the completion of each assay, a CO₂ pulse was delivered to thesensillum to test whether VUAA1 affected the CpA neuron; in contrast tothe responsiveness of the AgORco-expressing CpB/C neurons, CpA activitywas unchanged in the presence of vehicle and/or VUAA1 (FIGS. 6A, B, D).These data demonstrate that VUAA1 can specifically activateAgORco-expressing neurons in vivo. Moreover, VUAA1's ability to activateAgORco-expressing cells in vivo demonstrates that AgORco is anaccessible biological target, which is not directly obscured by otherproteins or cofactors involved in olfactory signal transduction. Assuch, VUAA1-mediated modulation serves as a proof-of-conceptdemonstration that AgORco is a viable target for the development ofbehaviorally disruptive olfactory compounds (BDOCs) that could fostermalaria reduction programs.

While the inventors cannot rule out an eventual identification, there iscurrently no evidence to support the existence of naturally-occurringAgORco ligands, which suggests that AgORco lacks a typical orthostericbinding site common to other ligand-gated ion channels. Without a moreadvanced structural analysis of AgORco, it is difficult to postulate asto the mechanism of VUAA1 gating, and whether it is acts in a mannerakin to canonical OR-dependent activation of the heteromeric OR complex.However, it is clear that AgORco is ionotropic, ligand-gated ionchannel.

In order to address whether VUAA1 as a non-volatile could also evokebehavioral responses from An. gambiae, the inventors tested behavioralresponses to VUAA1 on individual larval stage mosquitoes by adaptationof an olfactory based bioassay recently developed at Vanderbilt(described in Liu et al., 2010). These methods are non-invasive, andeasily executed using a fully automated setup with an Ethovision®camera/software system (Noldus Information Technology to quantify theoverall movement of mosquito larvae in response to uniformconcentrations of chemicals. In these assays, increased overall movementcan be interpreted as an aversive response (akin to agitation) that areconsistent with those evoked by commercially available insect repellentssuch as DEET.

With regard to VUAA1, these studies (FIG. 9) indicate that VUAA1 evokesrobust responses from An. gambiae larvae that are consistent withrepellency and the threshold of larval responses to VUAA1 (third througheighth bars from left) are 5 orders of magnitude lower than those toDEET (ninth through eleventh bars from left bars). Furthermore,gene-silencing studies using siRNAs directed against AgORco demonstratethat larval sensitivity to VUAA are dependent upon AgORco expression.Here larvae treated with AgOrco siRNAs (which reduce AgORco mRNA levels40-fold) no longer respond to VUAA1 stimuli while larvae receiving mockor non-specific siRNA control injection retain their high sensitivity toVUAA1.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and/or methods and in the steps or in the sequence of stepsof the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

IX. REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference:

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1. A method of disrupting odor-sensing behavior in an organism having anORco ion channel, comprising exposing the organism to a compound:

or a salt thereof.
 2. The method of claim 1, wherein the organism is aninsect.
 3. The method of claim 1, wherein the organism is a crop pest.3. The method of claim 1, wherein the organism is an airborne insect. 4.The method of claim 1, wherein the organism is a blood-sucking insect.5. The method of claim 1, wherein the organism is of the suborderIxodida.
 6. The method of claim 1, wherein the organism is of the orderDiptera.
 7. The method of claim 1, wherein the organism is of the orderLepidoptera.
 8. A method of agonizing an ORco ion channel, comprisingexposing the ORco ion channel to a compound:

or a salt thereof.
 9. The method of claim 8, wherein the ORco ionchannel is in vitro.
 10. The method of claim 9, wherein the ORco ionchannel is in a cultured cell.
 11. The method of claim 8, wherein theORco ion channel is in vivo.
 12. The method of claim 9, wherein the ORcoion channel is in an insect.
 13. A method of disrupting odor-sensingbehavior in an insect, comprising exposing the insect to a compound:

or a salt thereof.
 14. The method of claim 13, wherein the insect is amosquito.
 15. The method of claim 13, wherein the insect is a crop pest.16. The method of claim 13, wherein the insect is an airborne insect.17. The method of claim 13, wherein the insect is a blood-suckinginsect.
 18. The method of claim 13, wherein the insect is of thesuborder Ixodida.
 19. The method of claim 13, wherein the insect is ofthe order Diptera.
 20. The method of claim 13, wherein the insect is ofthe order Lepidoptera.